Diversity and plant growth promoting ability of rice root-associated bacteria in Burkina-Faso and cross-comparison with metabarcoding data.

Plant-associated bacteria are essential partners in plant health and development. In addition to taking advantage of the rapid advances recently achieved in high-throughput sequencing approaches, studies on plant-microbiome interactions require experiments with culturable bacteria. A study on the rice root microbiome was recently initiated in Burkina Faso. As a follow up, the aim of the present study was to develop a collection of corresponding rice root-associated bacteria covering maximum diversity, to assess the diversity of the obtained isolates based on the culture medium used, and to describe the taxonomy, phenotype and abundance of selected isolates in the rice microbiome. More than 3,000 isolates were obtained using five culture media (TSA, NGN, NFb, PCAT, Baz). The 16S rRNA fragment sequencing of 1,013 selected isolates showed that our working collection covered four bacterial phyla (Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes) and represented 33% of the previously described diversity of the rice root microbiome at the order level. Phenotypic in vitro analysis of the plant growth promoting capacity of the isolates revealed an overall ammonium production and auxin biosynthesis capacity, while siderophore production and phosphate solubilisation were enriched in Burkholderia, Ralstonia, Acinetobacter and Pseudomonas species. Of 45 representative isolates screened for growth promotion on seedlings of two rice cultivars, five showed an ability to improve the growth of both cultivars, while five others were effective on only one cultivar. The best results were obtained with Pseudomonas taiwanensis ABIP 2315 and Azorhizobium caulinodans ABIP 1219, which increased seedling growth by 158% and 47%, respectively. Among the 14 best performing isolates, eight appeared to be abundant in the rice root microbiome dataset from previous study. The findings of this research contribute to the in vitro and in planta PGP capacities description of rice root-associated bacteria and their potential importance for plants by providing, for the first time, insight into their prevalence in the rice root microbiome.

Paraburkholderia phytofirmans PsJN colonization of rice endosphere triggers an atypical transcriptomic response compared to rice native Burkholderia s.l. endophytes.

The plant microbiome has recently emerged as a reservoir for the development of sustainable alternatives to chemical fertilizers and pesticides. However, the response of plants to beneficial microbes emerges as a critical issue to understand the molecular basis of plant-microbiota interactions. In this study, we combined root colonization, phenotypic and transcriptomic analyses to unravel the commonalities and specificities of the response of rice to closely related Burkholderia s.l. endophytes. In general, these results indicate that a rice-non-native Burkholderia s.l. strain, Paraburkholderia phytofirmans PsJN, is able to colonize the root endosphere while eliciting a markedly different response compared to rice-native Burkholderia s.l. strains. This demonstrates the variability of plant response to microbes from different hosts of origin. The most striking finding of the investigation was that a much more conserved response to the three endophytes used in this study is elicited in leaves compared to roots. In addition, transcriptional regulation of genes related to secondary metabolism, immunity, and phytohormones appear to be markers of strain-specific responses. Future studies need to investigate whether these findings can be extrapolated to other plant models and beneficial microbes to further advance the potential of microbiome-based solutions for crop production.

Differential Genetic Strategies of Burkholderia vietnamiensis and Paraburkholderia kururiensis for Root Colonization of Oryza sativa subsp. japonica and O. sativa subsp. indica, as Revealed by Transposon Mutagenesis Sequencing.

Burkholderia vietnamiensis LMG10929 and Paraburkholderia kururiensis M130 are bacterial rice growth-promoting models. Besides this common ecological niche, species of the Burkholderia genus are also found as opportunistic human pathogens, while Paraburkholderia species are mostly environmental and plant associated. In this study, we compared the genetic strategies used by B. vietnamiensis and P. kururiensis to colonize two subspecies of their common host, Oryza sativa subsp. japonica (cv. Nipponbare) and O. sativa subsp. indica (cv. IR64). We used high-throughput screening of transposon insertional mutant libraries (Tn-seq) to infer which genetic elements have the highest fitness contribution during root surface colonization at 7 days postinoculation. Overall, we detected twice more genes in B. vietnamiensis involved in rice root colonization than in P. kururiensis, including genes contributing to the tolerance of plant defenses, which suggests a stronger adverse reaction of rice toward B. vietnamiensis than toward P. kururiensis. For both strains, the bacterial fitness depends on a higher number of genes when colonizing indica rice compared to japonica. These divergences in host pressure on bacterial adaptation could be partly linked to the cultivars' differences in nitrogen assimilation. We detected several functions commonly enhancing root colonization in both bacterial strains, e.g., Entner-Doudoroff (ED) glycolysis. Less frequently and more strain specifically, we detected functions limiting root colonization such as biofilm production in B. vietnamiensis and quorum sensing in P. kururiensis. The involvement of genes identified through the Tn-seq procedure as contributing to root colonization, i.e., ED pathway, c-di-GMP cycling, and cobalamin synthesis, was validated by directed mutagenesis and competition with wild-type (WT) strains in rice root colonization assays. IMPORTANCEBurkholderiaceae are frequent and abundant colonizers of the rice rhizosphere and interesting candidates to investigate for growth promotion. Species of Paraburkholderia have repeatedly been described to stimulate plant growth. However, the closely related Burkholderia genus includes both beneficial and phytopathogenic species, as well as species able to colonize animal hosts and cause disease in humans. We need to understand to what extent the bacterial strategies used for the different biotic interactions differ depending on the host and if strains with agricultural potential could also pose a threat toward other plant hosts or humans. To start answering these questions, we used in this study transposon sequencing to identify genetic traits in Burkholderia vietnamiensis and Paraburkholderia kururiensis that contribute to the colonization of two different rice varieties. Our results revealed large differences in the fitness gene sets between the two strains and between the host plants, suggesting a strong specificity in each bacterium-plant interaction.

Genetic Diversity of Type 3 Secretion System in Burkholderia s.l. and Links With Plant Host Adaptation.

Burkholderia sensu lato species are prominent for their diversity of hosts. The type 3 secretion system (T3SS) is a major mechanism impacting the interactions between bacteria and eukaryotic hosts. Besides the human pathogenic species Burkholderia pseudomallei and closely affiliated species, the T3SS has received little attention in this genus as in taxonomically and evolutionary close genera Paraburkholderia, Caballeronia, Trinickia, and Mycetohabitans. We proceeded to identify and characterize the diversity of T3SS types using the genomic data from a subset of 145 strains representative of the species diversity found in the Burkholderia s.l. group. Through an analysis of their phylogenetic distribution, we identified two new T3SS types with an atypical chromosomal organization and which we propose to name BCI (Burkholderia cepacia complex Injectisome) and PSI (Paraburkholderia Short Injectisome). BCI is the dominant T3SS type found in Burkholderia sensu stricto (s.s.) species and PSI is mostly restricted to the Paraburkholderia genus. By correlating their distribution with the ecology of their strains of origin, we propose a role in plant interaction for these T3SS types. Experimentally, we demonstrated that a BCI deficient B. vietnamiensis LMG10929 mutant was strongly affected in its rice colonization capacity.

Monitoring of Rice Transcriptional Responses to Contrasted Colonizing Patterns of Phytobeneficial Burkholderia s.l. Reveals a Temporal Shift in JA Systemic Response.

In the context of plant-pathogen and plant-mutualist interactions, the underlying molecular bases associated with host colonization have been extensively studied. However, it is not the case for non-mutualistic beneficial interactions or associative symbiosis with plants. Particularly, little is known about the transcriptional regulations associated with the immune tolerance of plants towards beneficial microbes. In this context, the study of the Burkholderia rice model is very promising to describe the molecular mechanisms involved in associative symbiosis. Indeed, several species of the Burkholderia sensu lato (s.l.) genus can colonize rice tissues and have beneficial effects; particularly, two species have been thoroughly studied: Burkholderia vietnamiensis and Paraburkholderia kururiensis. This study aims to compare the interaction of these species with rice and especially to identify common or specific plant responses. Therefore, we analyzed root colonization of the rice cultivar Nipponbare using DsRed-tagged bacterial strains and produced the transcriptomes of both roots and leaves 7 days after root inoculation. This led us to the identification of a co-expression jasmonic acid (JA)-related network exhibiting opposite regulation in response to the two strains in the leaves of inoculated plants. We then monitored by quantitative polymerase chain reaction (qPCR) the expression of JA-related genes during time course colonization by each strain. Our results reveal a temporal shift in this JA systemic response, which can be related to different colonization strategies of both strains.